Formulations of bendamustine

ABSTRACT

Methods of treatment using bendamustine formulations designed for small volume intravenous administration are disclosed. The methods conveniently allow shorter administration time without the active ingredient coming out of solution as compared to presently available formulations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of copending U.S. application Ser.No. 15/908,075, filed Feb. 28, 2018, which is a continuation of U.S.application Ser. No. 15/432,378, filed Feb. 14, 2017, now U.S. Pat. No.10,052,385, which is a continuation of U.S. application Ser. No.14/857,064, filed Sep. 17, 2015, now U.S. Pat. No. 9,597,397, which is acontinuation of U.S. application Ser. No. 14/714,578, filed May 18,2015, now U.S. Pat. No. 9,144,568, which is a continuation of U.S.application Ser. No. 13/838,090, filed Mar. 15, 2013, now U.S. Pat. No.9,034,908, which claims the benefit of priority from U.S. ProvisionalPatent Application No. 61/613,173, filed Mar. 20, 2012, and 61/669,889,filed Jul. 10, 2012, the disclosures of each of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Bendamustine is used in the treatment of a number of cancers includingleukemias, Hodgkin's disease and multiple myelomas. Bendamustine(present as the HCl salt) is the active ingredient of the commercialproduct Treanda™, a lyophilized powder for reconstitution. Currentlabeling requirements call for the reconstituted product to beimmediately (within 30 minutes) diluted into 500 mL of parenterallyacceptable diluents such as 0.9% saline (normal saline) or 2.5%dextrose/0.45% saline and administered as part of an intravenousinfusion delivering 100 mg/m² over 30 minutes or 120 mg/m² over 60minutes. The diluted admixture may be stored at 2-8° C. for up to 24hours, or 3 hours at room temperature (15-30° C.); administration mustbe completed within this period due to limited chemical stability inaqueous solutions.

Solubility limitations at 2-8° C. with currently approved and/oravailable formulations are believed to prevent current formulations frombeing administered in smaller more concentrated infusion volumes up toabout 150 ml; at volumes below 150 ml, solubility is not sufficient evenat 25° C. Side effects associated with extravasation and local erythema,swelling and pain at the injection site also dictate that the infusionbe as dilute as possible. Therefore, precautions are taken to avoidextravasation, including monitoring of the intravenous infusion site forredness, swelling, pain, infection, and necrosis during and afteradministration of bendamustine. Higher infusion volume and longerinfusion times, however, are associated with many drawbacks. Forexample, after reconstitution, the current product has a short period ofstability, degradation of the drug occurs from the time ofreconstitution until the entire large volume infusion has beencompletely administered. The current label for Treanda™ thereforeinstructs that the admixture should be prepared as close as possible tothe time of patient administration, and that administration of Treanda™must be completed within the durations indicated above. From patientcomfort and nursing administration points of view, higher infusionvolumes and long infusion times are undesirable. Higher infusion volumesmay be associated with higher likelihood of weight gain and edema.Shorter infusion times and smaller infusion volumes result in a betterquality of life experience for the patient by reducing the overall“stress” to the patient and reducing the time spent in the infusionclinic. Shorter infusion times (and smaller volumes) also reduce thepotential extravasation (and shorten the patient monitoring timerequired). It would be advantageous if the drug could be administered insmaller volumes and over shorter times. The present invention addressesthese needs.

SUMMARY OF THE INVENTION

In a first aspect of the invention there are provided methods oftreating or preventing cancer or malignant disease in a subject such asa human. The methods include parenterally administering a volume ofabout 325 ml or less of a liquid composition containing:

a) from about 0.05 to about 12.5 mg/ml of bendamustine or apharmaceutically acceptable salt thereof;

b) a solubilizer comprising polyethylene glycol and propylene glycol;and optionally

c) a parenterally acceptable diluent;

over a substantially continuous period of less than or equal to about 30minutes to a subject in need thereof.

In alternative aspects of the invention there are provided methods oftreating or preventing a bendamustine-responsive condition in a subjectsuch as a human. In a first embodiment the methods include administeringless than or equal to 325 ml of a liquid composition which contains

Ingredient Concentration Range (mg/ml) Bendamustine HCl 0.05 to 1.6Solubilizer 1 propylene glycol 0.30 to 6.5 Solubilizer 2 PEG 400  3.3 to65 Monothioglycerol 0.02 to 0.35 NaOH  0.0 to 0.01over a substantially continuous period of less than or equal to about 30minutes to a subject in need thereof.

In a related second embodiment of this aspect of the invention, themethods include administering less than or equal to 325 ml of a liquidcomposition which contains

Ingredient Concentration Range (mg/ml) Bendamustine HCl 1.1 to 12.5Solubilizer 1 propylene glycol 4.5 to 51 Solubilizer 2 PEG 400  45 to500 Monothioglycerol 0.2 to 2.5 NaOH 0.0 to 0.04over a substantially continuous period of less than or equal to about 30minutes to a subject in need thereof.

The methods of the present invention take advantage of the fact that theconcentration of the bendamustine HCl is below the room temperaturesolubility limit of the vehicle into which it is placed. As a result,the bendamustine does not precipitate during administration to thepatient thereby substantially avoiding the side effects which wouldotherwise occur during small volume administration of therapeutic dosesof the drug. In addition, patients or subjects withbendamustine-responsive conditions can be treated using substantiallysmaller parenteral volumes which are well below the standard 500 mladministration volume.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of a stability analysis of bendamustine in varioussolvents at 25° C.

FIG. 2 is a graph of a stability analysis of beridamustine in varioussolvents at 5° C.

FIG. 3 is a graph of bendamustine purity, over time, in 99% propyleneglycol, at 5° C. and at 25° C.

FIG. 4 shows the mean+standard deviation concentration-versus-timeprofiles of bendamustine in male Cynomolgus monkeys (N=4) administeredsingle 3 mg/kg bolus intravenous doses of bendamustine hydrochloride in3 different formulations.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. In the event that there is aplurality of definitions for a term herein, those in this sectionprevail unless stated otherwise.

In a first aspect of the invention there are provided methods oftreating or preventing cancer or malignant disease in a subject orpatient who is preferably a human. The methods generally includeparenterally administering a volume of about 325 ml or less of a liquidcomposition containing:

a) from about 0.05 to about 12.5 mg/ml of bendamustine HCl or apharmaceutically acceptable salt thereof, the HCl salt being preferred;

b) a solubilizer comprising polyethylene glycol and propylene glycol;and optionally

c) a parenterally acceptable diluent;

over a substantially continuous period of less than or equal to about 30minutes to a subject in need thereof.

The solubilizer portion of the formulation preferably includes fromabout 0.3 to about 45% volume polyethylene glycol (PEG) and from about0.03 to about 5% volume propylene glycol (PG), as calculated on thebasis of the total or final volume administered. Stated alternatively,the final concentration of the PEG generally ranges from about 3 toabout 500 mg/ml, while the final concentration of the PG generallyranges from about 0.5 to about 51 mg/ml. Within these general ranges,certain aspects of the invention include concentration ranges for thePEG of from about 45 to about 500 mg/ml or from about 3.3 to about 63.3mg/ml; and for the PG ranges of from about 4.7 to about 50.6; or fromabout 0.02 to about 6.5 mg/ml.

In some aspects of the invention, the bendamustine is administeredintravenously as part of an intravenous infusion. Contemplated infusionvolumes are preferably less than 325 ml with volumes such as about 250ml, 100 ml, and 50 ml, with each volume varying about +/−10% or +/−15%being preferred in some embodiments. In alternative aspects of theinvention, the intravenous administration volume is suitable for IVbolus administration and may also include an amount of pharmaceuticallyacceptable diluent such as normal saline or one of the other diluentsdescribed herein which does not cause the solubility of the vehicle tofall below the concentration of the bendamustine. Stated alternatively,the final concentration of the bendamustine will be below the solubilityof the combination vehicle containing the mixture of propylene glycoland PEG and diluent. While most aspects of the invention are describedin the context of administering less than about 325 ml including allvehicle ingredients, excipients, etc., it should be appreciated thatvolumes as low as a few milliliters, e.g. about 2, can be used so longas the vehicle includes sufficient solubilizers to preserve thesolubility of the bendamustine therein during administration to thepatient.

For purposes of the present invention, the word “about” when used tomodify infusion volumes or concentrations shall be understood to includevalues which may vary by amounts of about +/−10% or 15%. In certainembodiments where the infusion volume is about 50 ml, the concentrationof the bendamustine HCl or other pharmaceutically acceptable saltthereof is preferably from about 0.5 to about 5.6 mg/ml. In embodimentswhere the infusion volume is about 100 ml, the concentration of thebendamustine HCl or other pharmaceutically acceptable salt thereof canbe preferably from about 0.1 to about 3.2 mg/ml. Similarly, in someaspects of the invention where the infusion volume is about 250 ml, theconcentration of the bendamustine HCl or other pharmaceuticallyacceptable salt thereof is from about 0.05 to about 1.4 mg/ml.

The solubilizer is preferably a mixture of polyethylene glycol,hereinafter “PEG” and propylene glycol, hereinafter “PG”. Thesolubilizer can also optionally include an antioxidant such asmonothioglycerol. The amount of antioxidant included is a formulationstabilizing amount, which, in the case of monothioglycerol ranges fromabout 2 to about 10 mg/ml. The PEG preferably has a molecular weight ofabout 400, i.e. PEG 400. Other molecular weight PEG's known to those ofordinary skill can be included if desired in alternative embodiments.

Certain aspects of the invention call for the ratio of the PEG to PGfound in the solubilizer to be about 90:10. In alternative aspects, theratio of the PEG to PG is about 85:15.

In some aspects of the invention, the total amount of solubilizer, i.e.blend of PEG and PG, included in infusion volumes of about 100-115 ml isfrom about 0.5 to about 26.5% vol.; while amounts of from about 0.2 toabout 5% vol. for the solubilizer are preferably included in infusionvolumes of about 250-265 ml; with solubilizer amounts of from about 2.0to about 22.4% vol. included in infusion volumes of about 50-65 ml.

Since the solubilizer is a blend, the amount of PEG and PG in variousvolumes (calculated as % vol.) can be as follows:

Solubilizer 50 ml 100 ml 250 ml PEG 20.12 11.33 4.9 PG 2.24 1.26 0.54

In some preferred embodiments, the methods of the invention areadvantageously carried out using bendamustine HCl containingcompositions administered as small volume infusions with volumes ofabout 50 ml or about 100 ml or about 250 ml. Such smaller volumes allowthe drug to be administered over a time period of about 10 minutes orless as part of an intravenous infusions containing a volume of about 50ml; about 15 minutes or less as part of an intravenous infusionscontaining a volume of about 100 ml or when volumes of about 250 ml areinfused, the IV infusion is administered over a time period of about 30minutes or less. Depending upon the amount of drug administered, the IVbolus volumes containing sufficient amount of the drug will be less than50 ml, with amounts of about 10 or 15 to 30 ml being sufficient.

The infusible compositions in many aspects of the invention will alsopreferably include the parenterally acceptable diluents such as waterfor injection (WFI), 0.9% saline (normal saline, preferred), 0.45%saline (half normal saline) or 2.5% dextrose/0.45% saline. Formulationswell suited for carrying out the methods described herein are alsodescribed in commonly assigned U.S. patent application Ser. No.13/016,473, filed Jan. 28, 2011, and Ser. No. 13/767,672 filed Feb. 14,2013, the contents of which are incorporated herein by reference. Asreviewed in the '672 patent application, some preferred bendamustineformulations can also include a minor amount of a pH adjuster such assodium formate, sodium phosphate, potassium hydroxide, phosphoric acidor, preferably, sodium hydroxide.

In an alternative embodiment of the invention, the bendamustineformulations used in the methods described herein can be one or more ofthose described in U.S. Pat. Nos. 8,344,006 and 8,076,366; and USPublished Patent Application Nos. 2013/0041004; 2012/0071532;2010/0216858; 2006/0159713; and 2013/0041003, the contents of each ofwhich are incorporated herein by reference. It being understood that thevehicle into which the bendamustine HCl is placed will have sufficientbendamustine solubility which exceeds the concentration of the drugincluded therein.

For example, U.S. Published Patent Application No. 2010/0216858discloses a composition comprising: (a) bendamustine, (b) a chargedcyclopolysaccharide, and (c) a stabilizing agent having a chargeopposite to that of the cyclopolysaccharide.

Preferably, the proportion of active ingredient to cyclopolysaccharide,by weight, is between about 1:12,500 and about 1:25; is more preferablybetween about 1:5,000 and about 1:50; is even more preferably betweenabout 1:2,500 and about 1:75 and most preferably between about 1:1,500and 1:100.

The stabilizing agent is typically present in a weight ratio to thecyclopolysaccharide of between about 5:1 and about 1:1000; preferably ofbetween about 2:1 and about 1:200.

Cyclopolysaccharides

The cyclopolysaccharides which may employed in the practice of thisinvention include cyclodextrins, cyclomannins, cycloaltrins,cyclofructins and the like. In general, cyclopolysaccharides comprisingbetween 6 and 8 sugar units are preferred.

Among the preferred cyclopolysaccharides which may be employed arecyclodextrins.

Cyclodextrins are cyclic oligo-1-4-alpha-D-glucopyranoses comprising atleast 6 sugar units. The most widely known are cyclodextrins containingsix, seven or eight sugar units. Cyclodextrins containing six sugarunits are known as alpha-cyclodextrins, those containing seven sugarunits are known as beta-cyclodextrins and those consisting of eightsugar units are known as gamma-cyclodextrins. Particularly preferredcyclopolysaccharides are beta-cyclodextrins.

The cyclopolysaccharides employed are modified with one or morechargeable groups. Such chargeable groups may be anionic, in which casethe stabilizing agent is cationic; or such charged groups may becationic, in which case the stabilizing agent is anionic. Preferredanionic groups include carboxyl, sulfonyl and sulphate groups; whilepreferred cationic groups include quarternary ammonium groups.

As employed herein the term “charged cyclopolysaccharide” refers to acyclopolysaccharide having one or more of its hydroxyl groupssubstituted or replaced with a chargeable moiety. Such moiety may itselfbe a chargeable group (e.g., such as a sulfonyl group) or it maycomprise an organic moiety (e.g., a C₁-C₆ alkyl or C₁-C₆ alkyl ethermoiety) substituted with one or more chargeable moieties. Preferredsubstituted cyclopolysaccharides include, but are not limited to,sulfobutyl ether beta-cyclodextrin, beta-cyclodextrin substituted with2-hydroxy-N,N,N-trimethylpropanammonium,carboxymethylated-beta-cyclodextrin, O-phosphated-beta-cyclodextrin,succinyl-(2-hydroxy)propyl-beta-cyclodextrin,sulfopropylated-beta-cyclodextrin,heptakis(6-amino-6-deoxy)beta-cyclodextrin,O-sulfated-beta-cyclodextrin, and6-monodeoxy-6-mono(3-hydroxy)propylamino-b-cyclodextrin; with sulfobutylether beta-cyclodextrin being particularly preferred.

Cationic Stabilizing Agents

In those embodiments wherein the cyclopolysaccharide is modified withanionic groups, the stabilizing agent is selected from cationic agents,or from polycationic compounds. Cationic agents which may be employedinclude primary amines, secondary amines, tertiary amines or quaternaryammonium compounds, such as N-alkyl-N,N-dimethylamines,N-alkyl-N,N-diethylamines, N-alkyl-N—N-diethanoloamines,N-alkylmorpholine, N-alkylpiperidine, N-alkylpyrrolidine,N-alkyl-N,N,N-trimethylammonium, N,N-dialkyl-N,N-dimethylammonium,N-alkyl-N-benzyl-NN-diimethylammonium, N-alkyl-pyridinium,N-alkyl-picolinium, alkylamidomethylpyridinium, carbalkoxypyridinium,N-alkylquinolinium, N-alkylisoquinolinium, N,N-alkylmethylpyrollidinium,and 1-alkyl-2,3-dimethylimidazolium. Particularly preferred cationicadjuvants include sterically hindered tertiary amines, such asN-alkyl-N—N-diisopropylamine, N-alkylmorpholine, N-alkylpiperidine, andN-alkylpyrrolidine; and quaternary ammonium compounds such ascetylpyridinium chloride, benzyldimethyldodecylammonium chloride,dodecylpyridinium chloride, hexadecyltrimethylammonium chloride,benzyldimethyltetradecylammonium chloride,octadecyldimethylbenzylammonium chloride, and domiphen bromide.

Polycationic compounds such as oligo- or polyamines, or pegylated oligo-or polyamines may also be employed as the stabilizing agent. Preferredpolycationic compounds include oligoamines such as spermine, spermidin,putrescine, and cadaverine; polyamines: such as polyethyleneimine,polyspermine, polyputrescine, and polycadaverine; and pegylatedoligoamines and polyamines of the group listed above. Particularlypreferred is PI2080, polyethyleneimine 2000 conjugated with PEG 8000.

One preferred class of cationic stabilizing agents are polypeptidescomprising from about 5 to about 50, more preferably between about 6 andabout 20, amino acids; wherein at least about 50% of such amino acidscontain a positive charge. Most preferably, such charged amino acid isarginine. Particularly preferred members of this class of peptidesinclude arginine rich peptides comprising at least one block sequence of4 arginines. Another particularly preferred member of this class ofpeptides is protamine which has been digested with thermolysin(hereinafter referred to as Low Molecular Weight Protamine or “LMWP”).

Hydrophobically modified oligo- or polyamines may also be employed.Preferred stabilizing agent of this type include acetyl spermine, acetylpolyspermine, acetyl polyethyleneimine, butyryl spermine, butyrylpolyspermine, butyryl polyethyleneimine, lauroyl spermine, lauroylpolyspermine, lauroyl polyethyleneimine, stearoyl spermine, stearoylpolyspermine, and stearoyl polyethyleneimine.

In addition, cationic polysaccharides and synthetic polycationicpolymers may also be employed. Illustrative of such cationicpolysaccharides are chitosan, deacetylated chitosan, quaternizedcellulose, quaternized amylose, quaternized amylopectine, quaternizedpartially hydrolyzed cellulose, quaternized partially hydrolyzed amyloseand quaternized partially hydrolyzed amylopectine. Illustrative of suchsynthetic polycationic polymers are Polyquaternium 2(poly[bis(2-chloroethyl]ether-alt-1,3-bis[3-dimethylamino)propyl]-ureaquaternized); Polyquaternium 11(poly(1-vinylpyrrolidone-co-dimethylammonioethyl methacrylate)quaternized); Polyquaternium 16 and 44 (copolymer of vinylpyrrolidoneand quaternized vinylimidazole); and Polyquaternium 46 (copolymer ofvinylcaprolactam, vinylpyrrolidone and quaternized vinylimidazole).

Anionic Stabilizing Agents

In those embodiments wherein the cyclopolysaccharide is modified withcationic groups, the stabilizing agent is selected from anionic agents,or from polyanionic polymers.

Preferably, such anionic agent is selected from compounds comprising acarboxy-, sulfate-, sulfono-, phosphate-, or phosphono-group.

One class of anionic agents that may be employed are anionic surfactantssuch as sodium3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, sodiumN-lauroylsarcosinate, sodium dodecyl sulfate, sodiumdodecylbenzylsulfonate and the like.

Cationic polysaccharides may also be employed as the stabilizing agent.Illustrative of such compounds are chondroitin sulfate, dermatansulphate, kappa-carrageenan, iota-carrageenan, lambda-carrageenan,mu-carrageenan, xi-carrageenan, psi-carrageenan, tau-carrageenan,furcellaran, heparan sulphate, keratin, fucoidan, hyaluronic acid,alginic acid, poly(sulfonylbutylo)cellulose,poly(sulfonylpropylo)cellulose, poly(sulfonylpropylo)dextran,poly(sulfonylbutylo)dextran, poly(sulfonylbutylo)amylase andpoly(sulfonylpropylo)amylase.

The stabilizing agent may also be a polyanionic polymer selected frompolyacrylates, polymethacrylates, and their copolymers.

Excipients

The compositions of this invention may further contain pharmaceuticallyacceptable excipients, such as sugars, polyalcohols, soluble polymers,salts and lipids.

Sugars and polyalcohols which may be employed include, withoutlimitation, lactose, sucrose, mannitol, and sorbitol.

Illustrative of the soluble polymers which may be employed arepolyoxyethylene, poloxamers, polyvinylpyrrolidone, and dextran.

Useful salts include, without limitation, sodium chloride, magnesiumchloride, and calcium chloride.

Lipids which may be employed include, without limitation, fatty acids,glycerol fatty acid esters, glycolipids, and phospholipids.

Preparation

The composition of the invention may be prepared by the dissolution ofsolid bendamustine in an aqueous solution of the cyclopolysaccharide; orby mixing an aqueous solution of the cyclopolysaccharide with an aqueousstock solution of bendamustine. Such resulting mixture is vigorouslymixed and optionally subjected to the action of ultrasound waves toobtain an homogenous and equilibrated aqueous solution. When thecyclopolysaccharide is a cyclodextrin, it is preferred that the aqueoussolution of cyclodextrin used for the preparation of compositioncontains at least 4% of cyclodextrin; more preferably such solutioncontains at least 10% of cyclodextrin.

The stabilizing agent and excipient (if present) are preferablyintroduced to the composition by their addition to a pre-preparedaqueous homogenous and equilibrated solution of bendamustine with thecyclopolysaccharide. Such agents may be added either as pure substancesor as aqueous solutions and are preferably mixed employing gentleagitation.

Preferably, the final composition is filtered before use for injection.

The composition may be optionally freeze-dried to produce a solidmaterial suitable for dissolution in injection media before its use. Itis preferred that compositions comprising amines as stabilizing agentsare freeze dried prior to the addition of such stabilizing agent, withsuch agent being introduced into the composition after reconstitution,shortly before use.

In one embodiment the composition of this invention is prepared bymixing the components and incubation.

In another embodiment the composition of this invention is prepared bymixing the components and applying ultrasound to the mixture.

In another embodiment the composition of this invention is prepared bymixing the components, incubation, and freeze-drying the product.

In a preferred embodiment the composition of this invention is preparedby mixing the components, applying ultrasound to the mixture, andfreeze-drying the product.

The compositions of this invention demonstrate enhanced stability inaqueous solution and when introduced into plasma, both under in vitroand under in vivo conditions. Thus, such formulations will exhibit ahalf-life in plasma which is greater than that of non-formulatedbendamustine; which half-life may be extended by more than 50% andpreferably more than 100%.

In addition, the compositions of this invention exhibit unexpectedlyimproved activity against tumors relative to compositions comprisingbendamustine and a cyclopolysaccharide; as well as relative tobendamustine alone.

In other aspects, U.S. Pat. No. 8,344,006 discloses thatpharmaceutically acceptable liquid formulations of bendamustine, and thepharmaceutically acceptable salts thereof, in particular thehydrochloride salt, can be prepared by combining bendamustine, or thepharmaceutically acceptable salt thereof, with a polar aprotic solventor mixture of polar aprotic solvents. Polar, aprotic solvents are knownin the art and include, for example, 1-methyl-2-pyrrolidone,1,3-dimethyl-2-imidazolidinone, dimethylacetamide, dimethyl sulfoxide,acetone, tetrahydrofuran, 1,4-dioxane, acetonitrile, dimethyl formamide,propylene carbonate. See also, e.g., Florence Mottu, et al. Organicsolvents for pharmaceutical parenterals and embolic liquids: A review oftoxicity data, PDA J. Pharma. Sci. & Tech. vol 54, no. 6, 456-469(November-December 2000). Particularly preferred polar aprotic solventsinclude dimethylacetamide, dimethyl sulfoxide, and mixtures thereof.

Without wishing to be held to any particular theory, it is believed thatpolar, aprotic solvents are sufficiently non-nucleophilic towardsbendamustine such that polar aprotic solvent-bendamustine adducts do notform over the course of typical commercial storage conditions. Typicalcommercial storage conditions include time periods of, for example,about 30 days, about 90 days, about 180 days, and about 365 days (about1 month, about 3 months, about 6 months, and about 1 year). Typicalcommercial storage conditions also include temperatures of about 23° C.(ambient room temperature) and refrigerated temperatures below ambientroom temperature, for example, about 5° C. Preferably, the liquidformulations of the present invention are stored at refrigeratedtemperatures.

It has also been discovered that stable formulations of bendamustine canbe obtained by mixing a polar aprotic solvent, or a mixture of polaraprotic solvents, with a non-aqueous polar protic solvent or mixture ofnonaqueous polar protic solvents. Pharmaceutically acceptable nonaqueouspolar protic solvents are known in the art and include alkyl alcohols,for example, ethanol, ethylene glycol, propylene glycol, butyleneglycol, glycerin, polysorbates, for example TWEEN 20, TWEEN 40, andTWEEN 80, and cyclodextrins (such as hydroxypropyl-β-cyclodextrin),polyalkylene glycols, such as polyethylene glycol, polypropylene glycol,and polybutylene glycol, and primary amides such as niacinamide.

Such formulations will typically comprise 90% or less, by volume of theformulation, of the nonaqueous polar protic solvent. In other preferredembodiments, formulations will comprise between about 20% and about 85%,by volume of the formulation, of the nonaqueous polar protic solvent. Instill other embodiments, formulations will comprise between about 30%and about 70%, by volume of the formulation, of the nonaqueous polarprotic solvent. In most preferred embodiments, formulations willcomprise about 80%, about 67% or about 34%, by volume of theformulation, of the nonaqueous polar protic solvent.

Alternatively, formulations of the present invention will comprise 10moles per liter, or less, of the nonaqueous polar protic solvent.Preferably, formulations of the present invention will comprise betweenabout 4 moles per liter to about 9.5 moles per liter, of the nonaqueouspolar protic solvent. In certain embodiments, formulations will compriseabout 9.1 moles per liter of the nonaqueous polar protic solvent. Inother embodiments, formulations will comprise about 4.6 moles per liter,of the nonaqueous polar protic solvent.

While not wishing to be held to any particular theory, it is believedthat while nonaqueous polar protic solvents are of sufficientnucleophilicity to form potentially undesirable polar proticsolvent-bendamustine adducts, such adducts will not form during typicalcommercial storage if the concentration of the polar protic solvent iskept within the scope of the present invention.

Liquid formulations of the present invention are stable over the courseof a typical commercial storage period. As used herein, “stable” isdefined as no more than about a 10% loss of bendamustine under typicalcommercial storage conditions. Preferably, formulations of the presentinventions will have no more than about a 10% loss of bendamustine, morepreferably, no more than about a 5% loss of bendamustine, under typicalcommercial storage conditions.

Bendamustine converts to non-bendamustine products (i.e., “degrades”)upon exposure to certain nucleophiles, for example, water and alkyeneglycols such as propylene glycol. Exposure of bendamustine to water canproduce “HP1,” which is undesirable.

Another undesirable compound that bendamustine can convert to over timeis “BM1 dimer.”

Still another undesirable compound that bendamustine can convert to overtime is “DCE.”

Upon exposure to an alkylene glycol, for example, propylene glycol,esters of bendamustine can form, e.g., PG-1 and PG-2.

In preferred embodiments of the present invention, analysis offormulations of the present invention will exhibit 1.50% or less of DCE,as determined by HPLC analysis, after about 1 year (about 365 days) atabout 5° C. More preferably, the formulations will exhibit 1.0% or lessof DCE, as determined by HPLC analysis, after about 1 year (about 365days) at about 5° C. Even more preferably, the formulations will exhibit0.5% or less of DCE, as determined by HPLC analysis, after about 1 year(about 365 days) at about 5° C. Most preferably, the formulations willexhibit about 0.1% or less of DCE, as determined by HPLC analysis, afterabout 1 year (about 365 days) at about 5° C.

In other embodiments of the present invention, analysis of theformulations will exhibit about 0.4% or less of HP1, as determined byHPLC analysis, after about 1 year (about 365 days) at about 5° C.Preferably, the formulations will exhibit about 0.10% or less of HP1, asdetermined by HPLC analysis, after about 1 year (about 365 days) atabout 5° C.

In certain other embodiments of the present invention, analysis of theformulations will exhibit about 0.70% or less of BM1 dimer, asdetermined by HPLC analysis, after about 1 year (about 365 days) atabout 5° C. Preferably, the formulations will exhibit about 0.30% orless of dimer, as determined by HPLC analysis, after about 1 year (about365 days) at about 5° C. In most preferred embodiments, the formulationswill exhibit about 0.10% or less of BM1 dimer, as determined by HPLCanalysis, after about 1 year (about 365 days) at about 5° C.

In those embodiments of the present invention comprising alkylene glycolas the nonaqueous polar protic solvent, analysis of those formulationswill exhibit 1.5% or less of alkylene glycol esters of bendamustine, asdetermined by HPLC analysis, after about 1 year (about 365 days) atabout 5° C. For example, in those embodiments comprising propyleneglycol, analysis of those formulations will exhibit 1.5% or less ofpropylene glycol esters PG-1 and PG-2, as determined by HPLC analysis,after about 1 year (about 365 days) at about 5° C.

Analysis of the liquid formulations of the present invention can beperformed using techniques known in the art, including, for example,HPLC, gas chromatography, and NMR. After exposure to typical commercialstorage conditions, analysis of the formulations of the presentinvention will indicate that the formulation contains no less than about90% of the amount of bendamustine present prior to exposure to thestorage conditions. Preferably, analysis will indicate that theformulation contains no less than about 95% of the amount ofbendamustine present prior to exposure to the storage conditions.

In preferred embodiments of the present invention, analysis of theformulations of the present invention will indicate that the formulationcontains no less than about 90% of the amount of bendamustine presentprior to exposure to storage conditions that include temperatures ofabout 5° C. and time periods of about 30 days (about 1 month) to about365 days (about 1 year). Preferably, analysis of the formulations of thepresent invention will indicate that the formulation contains no lessthan about 90% of the amount of bendamustine present prior to exposureto storage conditions that include temperatures of about 5° C. and timeperiods of about 30 days (about 1 month), about 90 days (about 3months), and about 180 days (about 6 months). Preferably, analysis willindicate that the formulation contains no less than about 95% of theamount of bendamustine present prior to exposure to storage conditionsthat include temperatures of about 5° C. and time periods of about 30days (about 1 month) to about 365 days (about 1 year). More preferably,analysis will indicate that the formulation contains no less than about95% of the amount of bendamustine present prior to exposure to storageconditions that include temperatures of about 5° C. and time periods ofabout 30 days (about 1 month), about 90 days (about 3 months), and about180 days (about 6 months).

Formulations of the present invention can comprise pharmaceuticallyuseful concentrations of bendamustine, or a pharmaceutically acceptablesalt thereof. Useful concentrations include concentrations ranging fromabout 5 mg/mL to about 200 mg/mL. Preferably, the concentration ofbendamustine, or a pharmaceutically acceptable salt thereof, ranges fromabout 5 mg/mL to about 120 mg/mL. Preferred concentrations include about5 mg/mL, about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 40 mg/mL,about 50 mg/mL, about 60 mg/mL, about 100 mg/mL and about 200 mg/mL ofbendamustine, or a pharmaceutically acceptable salt thereof. Greaterthan 200 mg/ml of bendamustine, or a pharmaceutically acceptable saltthereof, for example, greater than about 300 mg/mL, are also within thescope of the present invention, as are saturated solutions ofbendamustine, or a pharmaceutically acceptable salt thereof.

In addition to comprising a polar aprotic solvent, or mixture of polaraprotic solvents, and optionally, a nonaqueous polar protic solvent, ormixture of solvents, formulations of the present invention may furthercomprise other pharmaceutically acceptable excipients. Pharmaceuticallyacceptable excipients are known in the art and include, for example,antioxidants (e.g., tocopherol (Vitamin E), ascorbic acid, methylparaben, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), andpropyl gallate), surfactants, (e.g., polysorbates (TWEEN 20, TWEEN 40,TWEEN 80)), lipids (e.g., dimyristoylphophatidylcholine (DMPC),Dimyristoylphosphatidylglycerol (DMPG), distearoylphophatidylglycerol(DSPG), fillers (e.g., mannitol), organic acids (e.g., citric acid,lactic acid, benzoic acid), hydrophilic polymers (e.g., polyethyleneglycols (PEG 300, PEG 400), complexing agents (e.g., niacinamide,nicotinic acid, creatine, cyclodextrins), and preservatives (e.g.,benzyl alcohol).

If desired, a sufficient amount of a concentrated, ready to use liquidformulation such one containing 25 mg/ml bendamustine HCl and alreadyadmixed with sufficient solubilizers can be transferred to a suitablefixed volume diluent container such as a bag containing 50, 100, 250 mlnormal saline or the like. Alternatively, lyophilized bendamustine HClcan be reconstituted, combined with sufficient solubilizer blends asdescribed herein and administered in accordance with the inventivemethods. In such embodiments, the actual amount delivered to the patientwill be slightly more than the diluent amount so as to allow for theaddition of the drug/solubilizer vehicle.

In some aspects of the invention, there are provided methods of treatingor preventing chronic lymphocytic leukemia (CLL). The small volumeinfusions can be given as part of any treatment protocol for whichbendamustine is included. Thus, the compositions described herein can beadministered as part of a poly-pharmaceutical treatment regimenaccording to known protocols with the exception that the concentratedbendamustine compositions described herein are administered in smallerinfusion volumes over significantly shorter administration periods. Forexample, some CLL treatment regimens can include administering thecompositions described herein intravenously as part of about 100 mlinfusions in about 20 minutes or less and more preferably in about 15minutes or less on days 1 and 2 of a 28 day cycle and repeating thecycle up to 6 times or longer if clinically appropriate. If 250 mlvolumes are used to deliver the bendamustine, the time of administrationis preferably about 30 minutes or less. If 50 ml volumes are used todeliver the bendamustine, the time of administration is preferably about10 minutes or less.

In spite of the smaller volumes, the amount of bendamustine HCladministered to the patient in need thereof per dose (infusion orotherwise) in some preferred embodiments is about 100 mg/m². In somealternative aspects of the invention, the amount of bendamustine HCladministered to the patient in need thereof as part of the 50, 100 or250 ml infusion is an amount sufficient to provide a dosage of 50 or 25mg/m². Additional administration dosages will be apparent to those ofordinary skill based upon clinical experience, patient need withoutundue experimentation.

In other aspects of the invention, there are methods of treating orpreventing the malignant disease of indolent B-cell non-Hodgkin'slymphoma. In these aspects, the composition is administeredintravenously as a 100 ml infusion in less than 20 minutes and morepreferably in about 15 minutes or less on days 1 and 2 of a 21 day cyclefor up to 8 cycles or longer if clinically appropriate. If 250 mlvolumes are used to deliver the bendamustine, the time of administrationis preferably about 30 minutes or less. If 50 ml volumes are used todeliver the bendamustine, the time of administration is preferably about10 minutes or less. The amount of bendamustine administered to thesubject is preferably about 120 mg/m², although in alternativeembodiments, the amount administered ranges from about 90 or 60 mg/m².As will be appreciated, further alternative dosage amounts will beapparent to those of ordinary skill based upon clinical experience,patient need without undue experimentation.

It will be appreciated by those skilled in the art that theabove-mentioned dosages calculated in mg/m² for purposes of body surfacearea (B S A) are consistent with the bendamustine HCl concentrationsalso described herein, e.g. 0.5 to 5.6 mg/ml. In the alternative, theinvention also contemplates IV bolus administration ofbendamustine-containing formulations in volumes which can beadministered via syringe, e.g. from a few milliliters up to about 50milliliters, with therapeutic amounts of the drug in a concentrationwhich does not exceed the vehicle solubility for the drug therein.

Further embodiments of the invention include methods of treating orpreventing a bendamustine-responsive condition in a subject such as ahuman. In a first embodiment, the methods include administering lessthan or equal to 325 ml of a liquid composition which contains

Ingredient Concentration Range (mg/ml) Bendamustine HCl 0.05 to 1.6 Solubilizer 1 propylene glycol 0.30 to 6.5  Solubilizer 2 PEG 400 3.3 to65  Monothioglycerol 0.02 to 0.35 NaOH  0.0 to 0.01over a substantially continuous period of less than or equal to about 30minutes to a subject in need thereof. More preferably, theadministration time is well below 30 minutes and the administration timewill decrease as the volume administered decreases.

Bendamustine formulations containing the above ingredients are capableof delivering approximately 25 mg of the drug as the HCl salt in volumesof pharmaceutically acceptable diluent ranging from about 325 ml down toabout 15 ml. For example, 1 ml of a bendamustine HCl ready to use liquidavailable from Eagle Pharmaceuticals containing

Ingredient Concentration (mg/ml) Bendamustine HCl 25 PG 103.2 PEG 4001013.4 Monothioglycerol 5 NaOH 0.08is combined with 300 ml of a normal saline diluent to provide a final IVinfusion containing 301 ml and a bendamustine final concentration of0.08 mg/ml.

One ml of the 25 mg/ml Eagle bendamustine HCl is diluted into additionaldiluent volumes as shown below:

Final Bendamustine Diluent Volume (ml) Final Volume (ml) Conc. (mg/ml)200 201 0.12 100 101 0.25 50 51 0.49 30 31 0.81 15 16 1.56

The measured solubility of the bendamustine HCl in thediluent/solubilizer combination (50 ml diluent plusl ml of 25 mg/mlbendamustine HCl and solubilizers, etc.) at room temperature was 10.5mg/ml using normal saline and 14.2 mg/ml using half normalsaline/dextrose. The solubility of the diluent/solubilizer combinationfar exceeded the bendamustine concentration, thus assuring the avoidanceof precipitated drug prior to or during administration. As will beappreciated by those of ordinary skill, as the concentration ofsolubilizers increases with respect to the total volume in smalladministration doses, the solubility of the bendamustine is maintained.

In a related second embodiment of this aspect of the invention, themethods include administering less than or equal to 325 ml of a liquidcomposition which contains

Ingredient Concentration Range (mg/ml) Bendamustine HCl  1.1 to 12.5Solubilizer 1 propylene glycol 4.5 to 51  Solubilizer 2 PEG 400  45 to500 Monothioglycerol 0.2 to 2.5 NaOH  0.0 to 0.04over a substantially continuous period of less than or equal to about 30minutes to a subject in need thereof. As was the case above, theadministration time will decrease with the decrease in volumeadministered.

Bendamustine formulations containing the above ingredients are capableof delivering approximately 360 mg of the drug as the HCl salt involumes of pharmaceutically acceptable diluent ranging from about 325 mldown to about 15 ml. As was the case above, the measured solubility ofthe bendamustine HCl in the diluent/solubilizer combination (1 mldrug+solubilizers, etc. and 50 ml diluent) at room temperature was 10.5mg/ml using normal saline and 14.2 mg/ml using half normalsaline/dextrose.

Instead of using only 1 ml of the above described Eagle 25 mg/mlbendamustine HCl ready to use liquid, 14.4 ml is combined with variousamounts of diluent.

Final Bendamustine Diluent Volume (ml) Final Volume (ml) Conc. (mg/ml)300 314.4 1.15 200 214.4 1.68 100 114.4 3.15 50 64.4 5.59 30 44.4 8.1115 29.4 12.24

In each case, the solubility of the diluent/solubilizer combinationexceeds the bendamustine concentration, thus assuring the avoidance ofprecipitated drug prior to or during administration.

EXAMPLES

The following examples serve to provide further appreciation of theinvention but are not meant in any way to restrict the effective scopeof the invention.

Example 1

The solubility of bendamustine HCl, obtained from two different sources,in 0.9% saline and 0.9% saline containing from different amounts of anon-aqueous solubilizer comprising a mixture of polyethylene glycol 400and propylene glycol (in the volume proportion of 90:10) with andwithout 5 mg/ml monothioglycerol was determined at both room temperature(22-23° C.) and at refrigerated temperature (5° C.). Essentially, anexcess of bendamustine HCl was added to solvents comprising of variousvolume percent of the non-aqueous solubilizer in 0.9% saline, andallowed to equilibrate with shaking for 30 minutes at room temperature,or for 24 hours at refrigerated temperature. At the end of theequilibration step, the suspensions were filtered through a 0.2 micronfilter to remove undissolved bendamustine, and the filtrate solutionsanalyzed for bendamustine HCl content using a HPLC assay; quantificationwas performed against a bendamustine HCl reference standard. Thesolubility data are presented in Table 1.

TABLE 1 Solubility of bendamustine HCl in 0.9% saline with variousamounts of non-aqueous solubilizer (90:10 PEG400:PG with and without 5mg/mL monothioglycerol (MTG)) Solubility of bendamustine HCl VolumeVolume % (mg/mL) in 90:10 PEG 400/PG % of of Non- Room temperature 5° C.Dilution Normal aqueous API API API Fold Saline Solubilizer Source ASource B* B Source A n/a 100.0 0.0 3.461 3.304 1.175 40 97.5 2.5 3.9873.889 nd 20 95.0 5.0 4.429 4.204 2.022 13.3 92.5 7.5 nd 4.742 nd 10 90.010.0 5.626 5.351 2.431 8 87.5 12.5 nd 5.825 nd 6.7 85.0 15.0 7.012 6.5542.900 5.7 82.5 17.5 nd 7.641 3.328 5 80.0 20.0 8.642 8.492 3.824 3.370.0 30.0 12.006 11.407 nd *solvent also contained 5 mg/mlmonothioglycerol nd = not determined; API = active pharmaceuticalingredient

Example 2

Bendamustine-containing compositions are prepared by adding 5 mg/ml ofthioglycerol to a mixture containing 90% polyethylene glycol 400 and 10%propylene glycol. As indicated in the Table 2 below, NaOH may be addedto the PEG in an amount sufficient to get apparent pH of greater than orequal to 6.5 as measured using the pH method outlined in the USPmonograph for polyethylene glycol (PEG). Bendamustine (BDM) is thenadded to the sample to a concentration of 10 mg/ml.

TABLE 2 Formulation   BDM-10 mg/mL Thioglycerol-5 mg/mL PEG 400:PG(90:10) qs to 1 mL BDM-10 mg/mL Thioglycerol-5 mg/mL PEG 400:PG (90:10)qs to 1 mL (PEG 400 Treated with NaOH)

The compositions are then admixed with normal saline based on the totaldose of bendamustine HCl, which in turn is based on the patient bodysurface area (B S A) and the dosing regimen (100 mg/m² for CLL and 120mg/m² for NLL; although dose modifications of 90, 60, 50, and 25 mg/m²are possible, only the highest two dosing regimens are considered forillustrative purposes, as these result in the highest concentration ofbendamustine during infusion). The 100 ml infusion is then made byadmixing the dose appropriate volume of the 10 mg/ml solution with a 100ml portion of normal saline to provide an infusible compositioncontaining the appropriate dose of bendamustine (as the HCl salt) in thefinal admixture, which can be administered intravenously over about 15minutes to a patient in need thereof.

As seen in Table 3, the concentrations of bendamustine (as HCl salt) andthe corresponding volume percent of non-aqueous component are well belowthe corresponding solubilities at both room temperature and refrigeratedtemperature as detailed in Table 1. For example, for a 2.0 m² (average)patient dosed at 120 mg/m2, the final concentration of bendamustine HClin a 100 ml admixture is 1.94 mg/mL. This is above the solubility ofbendamustine HCl at refrigerated storage conditions in the absence ofany non-aqueous components (1.175 mg/ml as shown in Table 1 for 100%normal saline), as would be the case with the currently approvedTreanda™ product, thereby precluding preparation and storage of a 100 mladmixture volume at refrigerated conditions. However, the use of thenon-aqueous bendamustine formulation described in this example resultsin the presence of 19.4% of the non-aqueous component in the finaladmixture, which improves the solubility to about 3.8 mg/mL (solubilityof 3.824 mg/mL at 2-8° C. with 20% non-aqueous component, as shown inTable 1). Therefore, the solubility with the non-aqueous formulation iswell above the final concentration (of bendamustine HCl) of 1.94 mg/mL,allowing preparation and storage of the 100 ml admixture at refrigeratedconditions. In this example, the room temperature solubilities in 100%normal saline and 80% normal saline (with 20% non-aqueous component) areabout 3.3 mg/ml and 8.5 mg/ml, respectively (see Table 1), which arealso well above the final concentration of 1.94 mg/ml. Therefore, 100 mladmixtures of the non-aqueous formulation described in the example mayalso be prepared and stored at room temperature. In addition, thenon-aqueous formulation of bendamustine described in this example atTable 2 may be diluted into smaller infusion volumes ranging from 250 mlor less, and stored at either room temperature or refrigeratedtemperature, with bendamustine continuing to remain in solution forextended periods of time as compared to currently availableformulations.

TABLE 3 Concentrations of bendamustine (BDM, as HCl salt) andcorresponding volume % of non-aqueous (NA) component in the finaladmixture, for volumes ranging from 100 ml to 250 ml For 10 mg/mLFormulation 100 mg/ 120 mg/ 100 mg/ 120 mg/ m² dose m² dose m² dose m²dose BDM % BDM % BDM % BDM % Conc NA Conc NA Conc NA Conc NA BSA (m²)(mg/ml) comp. (mg/ml) comp. (mg/ml) comp. (mg/ml) comp. Admix. Volume250 mL Admix. Volume 200 mL 1.00 0.38 3.8 0.46 4.6 0.48 4.8 0.57 5.71.25 0.48 4.8 0.57 5.7 0.59 5.9 0.70 7.0 1.50 0.57 5.7 0.67 6.7 0.70 7.00.83 8.3 1.75 0.65 6.5 0.77 7.7 0.80 8.0 0.95 9.5 2.00 0.74 7.4 0.88 8.80.91 9.1 1.07 10.7 2.25 0.83 8.3 0.97 9.7 1.01 10.1 1.19 11.9 2.50 0.919.1 1.07 10.7 1.11 11.1 1.30 13.0 2.75 0.99 9.9 1.17 11.7 1.21 12.1 1.4214.2 3.00 1.07 10.7 1.26 12.6 1.30 13.0 1.53 15.3 Admix. Volume 150 mLAdmix. Volume 100 mL 1.00 0.63 6.3 0.74 7.4 0.91 9.1 1.07 10.7 1.25 0.777.7 0.91 9.1 1.11 11.1 1.30 13.0 1.50 0.91 9.1 1.07 10.7 1.30 13.0 1.5315.3 1.75 1.04 10.4 1.23 12.3 1.49 14.9 1.74 17.4 2.00 1.18 11.8 1.3813.8 1.67 16.7 1.94 19.4 2.25 1.30 13.0 1.53 15.3 1.84 18.4 2.13 21.32.50 1.43 14.3 1.67 16.7 2.00 20.0 2.31 23.1 2.75 1.55 15.5 1.80 18.02.16 21.6 2.48 24.8 3.00 1.67 16.7 1.94 19.4 2.31 23.1 2.65 26.5

Example 3

The procedures of Example 2 are repeated except that the doseappropriate volume of the 10 mg/ml bendamustine solution is diluted into250 ml of normal saline. The final concentration of bendamustine in the250 ml volume container ranges from about 0.05 mg/ml to about 1.3 mg/ml.

Example 4

The approximately 100 ml bendamustine HCl infusion of Example 2 isadministered to a patient in about 15 minutes.

Example 5

Bendamustine-containing compositions may be prepared by adding 5 mg/mlof thioglycerol to 90% polyethylene glycol 400 and 10% propylene glycol.As indicated in the Table 4 below, NaOH may be added in an amountsufficient to get apparent pH of greater than or equal to 6.5 asmeasured using the pH method outlined in the USP monograph forpolyethylene glycol (PEG). Bendamustine is then added to the sample to aconcentration of 25 mg/ml as indicated in Table 4 below.

TABLE 4 Formulation BDM-25 mg/mL   Thioglycerol-5 mg/mL PEG 400:PG(90:10) qs to 1 mL BDM-25 mg/mL Thioglycerol-5 mg/mL PEG 400:PG (90:10)qs to 1 mL (PEG 400 Treated with NaOH)

The compositions are then admixed with normal saline based on the totaldose of bendamustine HCl, which in turn is based on the patient bodysurface area (B S A) and the dosing regimen (100 mg/m² for CLL and 120mg/m2 for NLL; although dose modifications of 90, 60, 50, and 25 mg/m²are possible, only the highest two dosing regimens are considered forillustrative purposes, as these result in the highest concentration ofbendamustine during infusion). Table 5 below provides the finalconcentration of bendamustine (as the HCl salt) in the final admixture,for volumes ranging from 250 ml to 50 ml.

As seen in Table 5, the concentrations of bendamustine (as HCl salt) andthe corresponding volume percent of non-aqueous component are well belowthe corresponding solubilities at room temperature as detailed in Table1, for all admixture volumes up to 50 ml. For example, for a 2.0 m²(average) patient dosed at 120 mg/m², the final concentration ofbendamustine HCl in a 50 ml admixture is 4.03 mg/ml. This is above thesolubility of bendamustine HCl at both refrigerated and room temperatureconditions in the absence of any non-aqueous components (1.175 mg/ml at2-8° C. and 3.304-3.461 mg/ml at room temperature, as shown in Table 1for 100% normal saline), as would be the case with the currentlyapproved Treanda product, thereby precluding preparation and storage ofa 50 ml admixture volume. However, the use of the non-aqueousbendamustine formulation described in this example results in thepresence of 16.1% of the non-aqueous component in the final admixture,which improves the room temperature solubility to about 6.5 mg/ml(solubility of 6.554 mg/ml and 7.012 mg/ml with 15% non-aqueouscomponent, as shown in Table 1). Therefore, the solubility with thenon-aqueous formulation is well above the final concentration (ofbendamustine HCl) of 4.03 mg/mL, allowing preparation and storage of the100 ml admixture at room temperature conditions. Therefore, thenon-aqueous formulation of bendamustine described in this example may bediluted into smaller infusion volumes ranging from 250 ml or less, withbendamustine continuing to remain in solution if maintained at roomtemperature. However, at refrigerated temperatures, the concentrationsof bendamustine (as HCl salt) and the corresponding volume percent ofnon-aqueous component exceed the corresponding solubilities as detailedin Table 1, for all admixture volumes equal to or below 150 ml. In thescenario above, the solubility at refrigerated conditions with 15%non-aqueous component has improved to 2.9 mg/ml but is still below thefinal concentration of 4.03 mg/ml. Therefore, 50 ml admixtures of thenon-aqueous formulation described in the example cannot be prepared andstored at refrigerated temperatures. However, for a 150 ml admixture,the final concentration of bendamustine HCl in this scenario is 1.5mg/ml with about 6.0% non-aqueous component, which is below thesolubility limit (of 2.022 mg/ml at 5% non-aqueous at 2-8° C.).Therefore, the non-aqueous formulation of bendamustine described in thisexample may be diluted into smaller infusion volumes ranging from 250 mlto 50 ml, and stored at only room temperature (but not refrigeratedtemperature), with bendamustine continuing to remain in solution. Forstorage at refrigerated temperatures, the minimum admixture volume thatcan be used is 150 ml or higher.

TABLE 5 Concentrations of bendamustine (BDM, as HCl salt) andcorresponding volume % of non-aqueous (NA) component in the finaladmixture, for volumes ranging from 100 ml to 250 ml For 25 mg/mLFormulation 100 mg/ 120 mg/ 100 mg/ 120 mg/ m² dose m² dose m² dose m²dose BDM % BDM % BDM % BDM % Conc NA Conc NA Conc NA Conc NA BSA (m²)(mg/ml) comp. (mg/ml) comp. (mg/ml) comp. (mg/ml) comp. Admix. Volume250 mL Admix. Volume 200 mL 1.00 0.39 1.6 0.47 1.9 0.49 2.0 0.59 2.31.25 0.49 2.0 0.59 2.3 0.61 2.4 0.73 2.9 1.50 0.59 2.3 0.70 2.8 0.73 2.90.87 3.5 1.75 0.68 2.7 0.81 3.3 0.85 3.4 1.01 4.0 2.00 0.78 3.1 0.92 3.70.96 3.8 1.15 4.6 2.25 0.87 3.5 1.04 4.1 1.08 4.3 1.28 5.1 2.50 0.96 3.81.15 4.6 1.19 4.8 1.42 5.7 2.75 1.05 4.2 1.25 5.0 1.30 5.2 1.55 6.2 3.001.15 4.6 1.36 5.4 1.42 5.7 1.68 6.7 Admix. Volume 150 mL Admix. Volume100 mL 1.00 0.65 2.6 0.78 3.1 0.96 3.8 1.15 4.6 1.25 0.81 3.2 0.96 3.81.19 4.8 1.42 5.7 1.50 0.96 3.8 1.15 4.6 1.42 5.7 1.68 6.7 1.75 1.11 4.51.33 5.3 1.64 6.5 1.94 7.7 2.00 1.27 5.1 1.50 6.0 1.85 7.4 2.19 8.8 2.251.42 5.7 1.68 6.7 2.06 8.3 2.44 9.7 2.50 1.56 6.3 1.85 7.4 2.27 9.1 2.6810.7 2.75 1.71 6.8 2.02 8.1 2.48 9.9 2.92 11.7 3.00 1.85 7.4 2.19 8.82.68 10.7 3.15 12.6 Admix. Volume 50 mL 1.00 1.85 7.4 2.19 8.8 1.25 2.279.1 2.68 10.7 1.50 2.68 10.7 3.15 12.6 1.75 3.07 12.3 3.60 14.4 2.003.45 13.8 4.03 16.1 2.25 3.81 15.3 4.44 17.8 2.50 4.17 16.7 4.84 19.42.75 4.51 18.0 5.22 20.9 3.00 4.84 19.4 5.59 22.4

Example 6

The hemolytic potential of the non-aqueous bendamustine formulationindicated in Table 4 (Example 5), when admixed with 250 ml and 100 ml ofnormal saline, was assessed. The hemolysis study was conducted at thehighest final bendamustine HCl concentrations expected at theseadmixture volumes, namely, for a 3.0 m² patient dosed at 120 mg/m². Atthis dosing, the final bendamustine HCl concentration for 250 ml and 100ml admixture volumes is 1.36 mg/ml and 3.15 mg/ml, respectively (Table5). Human whole blood (1 ml) was incubated at 37° C. for approximately30 minutes with admixed bendamustine HCl solutions at 1.4 mg/ml or 3.2mg/ml at blood to drug solution volumetric ratios of 1:2 and 1:1,respectively. These volumetric ratios correspond to infusion times of 15minutes and 10 minutes, respectively, for the 250 ml and 100 mladmixture volumes. A placebo of the bendamustine formulation (withoutthe active ingredient) was also evaluated at these concentrations andvolumetric ratios. A positive control (1% saponin solution), a negativecontrol (normal saline), and Treanda™ diluted in normal saline to thehighest concentration stated in the prescribing information (0.6 mg/ml)were included in the study. Following incubation and centrifugation ofthe samples, the plasma was harvested and hemolysis was evaluated byspectrophotometric analysis for hemoglobin in the supernatant. Theresults are summarized in Table 6. No hemolysis was observed with thenon-aqueous bendamustine formulation when diluted with saline at eitherconcentration or volumetric (blood:drug solution) ratios, or with thecorresponding placebo at comparable sample volumes; supernatants fromall samples were light yellow. In conclusion, no hemolytic effects areobserved with non-aqueous bendamustine formulations when diluted tosmaller volumes (100 to 250 ml) and infused in shorter times (10-15minutes) than current practice.

TABLE 6 Hemolytic Potential test results Hemoglobin^(a) Test SupernatantMixture (mg/dL) Result Color^(b) Tube No. Human blood plus: Test ArticleA (25 mg/mL, diluted to 3.2 mg/mL with saline)-100 ml admixture 1 NLight yellow  1 0 N Light yellow  2 1 N Light yellow  3 Test Article A(25 mg/mL, diluted to 1.4 mg/mL with saline)-250 ml admixture 2 N Lightyellow  4 1 N Light yellow  5 1 N Light yellow  6 Test Article A Vehicle(Placebo, diluted with 100 ml saline) 0 N Light yellow  7 1 N Lightyellow  8 0 N Light yellow  9 Test Article A Vehicle (Placebo, dilutedwith 250 ml saline) 1 N Light yellow 10 4 N Light yellow 11 1 N Lightyellow 12 Treanda ™ (5 mg/mL, diluted to 0.6 mg/mL with saline) 3 NLight yellow 43 4 N Light yellow 44 2 N Light yellow 45 Negative Control(normal saline) 9 N Yellow 55 5 N Yellow 56 3 N Yellow 57 PositiveControl (1% Saponin)^(c) 5949   P Red 58 5974   P Red 59 6386   P Red 60N = Negative, no hemolysis. NA = Not applicable. P = Positive,hemolysis. ^(a)Hemoglobin index of the mixture supernatants. ^(b)Plasmaseparated from whole blood plasma. ^(c)1% Saponin. Saponin is ahemolytic agent used to lyse erythrocytes.

Example 7

The local tolerance (intravenous (IV) and perivascular (PV)) of thenon-aqueous bendamustine-containing composition indicated in Table 4(Example 5), when admixed with 100 ml of normal saline and infused over10 minutes, was assessed. New Zealand White rabbits (3 males [IV] and 2males [PV]) received a single dose of bendamustine formulation (admixedwith 100 ml saline to a final concentration of 3.2 mg/ml bendamustineHCl) and corresponding placebo in the left and right ear, respectively.The formulation was administered as either intravenous infusion (5 mg/kgin 10 minutes), or perivascular injection (250 μl) to determine localtolerance. Treanda™ reconstituted and admixed with normal saline to afinal concentration of 0.6 mg/ml (the highest concentration stated inthe label) was also studied either as a 30 minute IV infusion (theshortest infusion time stated in the label), as well as perivascularinjection (250 μl). Animals were held for a 96 hour (post-dose)observation period. During the observation period, dermal scores wererecorded for all administration sites. At the end of the observationperiods, animals were euthanized and a macroscopic and microscopicexamination of both ears was performed. Parameters evaluated during thestudy were: viability, clinical observations, body weights, macroscopicobservations and microscopic pathology.

The results of the local tolerance study are summarized in Table 7 (inlife dermal observations) and Table 8 (microscopic pathology forperivascular administration).

In Life Dermal Observations:

As seen in Table 7, there was transient, dermal irritation in the formof slight to moderate erythema and moderate edema noted between 24 and72 hours post dose, in each of the groups receiving eitherbendamustine-containing formulations or placebo material intravenously.At 96 hours, irritation was limited to a few individual sites treatedwith test or placebo articles. Only a limited number of animals wereaffected, and there was no consistent pattern of irritation within adose group (either for test article or placebo). The bendamustineformulations were considered not to produce dermal irritation whenadministered intravenously.

Perivascular administration of bendamustine formulations (0.25 mlinjection volume) produced dermal irritation in all groups. Local signsof dermal irritation following perivascular administration were mostlycharacterized by slight (group 6—Treanda™) or slight to moderate (group7—non aqueous bendamustine formulation of example 5) erythema, andslight edema (groups 7). The severity of the irritation observedcorrelated with the dose and/or concentration of the test articleadministered, with placebo groups generally showing a lesser level ofirritation than the corresponding test-article formulation.

TABLE 7 Summary of in life dermal observations Dermal observationsduring 96 hour post dose period (incidence and most severe level oferythema and edema noted) Left ear (Bendamustine- Right containing ear(Placebo Material-Left ear/Right ear formulation) material) Intravenousadministration Group 1: Treanda ™ diluted to 0.6 — (2/3) Slight mg/ml insaline/Treanda Placebo- 500 ml admixture Group 2: Non-aqueousbendamustine — (1/3) formulation 25 mg/ml diluted to 3.2 Moderate mg/mlin saline/placebo (+saline)- 100 ml admixture Perivascularadministration Group 6: Treanda ™ diluted to 0.6 (1/2) Slight (1/2)Slight mg/ml in saline/Treanda ™ Placebo- 500 ml admixture Group 7:Non-aqueous bendamustine (2/2) Moderate (2/2) Slight formulation 25mg/ml diluted to 3.2 mg/ml in saline/placebo (+saline)- 100 ml admixture

Microscopic Pathology:

Intravenous administration of test articles/placebos was generally welltolerated; no test article related effects were observed. Perivascularadministration of bendamustine-containing formulations (includingTreanda™) was associated with dose and/or concentration related minimalto marked edema/collagen degeneration and mixed inflammation inperivascular tissues. The non-aqueous formulation of bendamustine (Group7) was nominally more severe in grade than Treanda™ (Group 6).

TABLE 8 Incidence and Average Severity of Microscopic Findings atPerivascular Sites Treanda ™ Non Aqueous (diluted Bendamustine to 0.6mg/ml)/ (diluted to 3.2 Treanda ™ mg/ml)/ Formulation Placebo PlaceboGroup number 6 7 No. Animals examined 2 2 RIGHT EAR (RE)-PlaceboIncidence (Average Severity)* RE Injection site Hemorrhage 0 (0.0) 0(0.0) Mixed Inflammation 0 (0.0) 0 (0.0) Edema/Collagen Degeneration 1(0.5) 0 (0.0) Degeneration/Inflammation, Vascular 0 (0.0) 0 (0.0) RE 2cm distal Edema/Collagen Degeneration 0 (0.0) 0 (0.0)Degeneration/Inflammation, Vascular 0 (0.0) 0 (0.0) RE 4 cm distalDegeneration/Inflammation, Vascular 0 (0.0) 0 (0.0) LEFT EAR (LE)-TestArticle Incidence (Average Severity) LE Injection site Hemorrhage 0(0.0) 0 (0.0) Mixed inflammation 0 (0.0) 2 (2.0) Edema/CollagenDegeneration 1 (1.0) 2 (1.5) Epidermis, Crust/Pustule, Erosion/ 0 (0.0)0 (0.0) Ulceration Degeneration/Inflammation, Vascular 1 (1.5) 1 (1.5)LE 2 cm distal Hemorrhage 0 (0.0) 0 (0.0) Mixed Inflammation 1 (0.5) 1(1.0) Edema/Collagen Degeneration 0 (0.0) 2 (2.0) Epidermis,Crust/Pustule, Erosion/ 0 (0.0) 0 (0.0) UlcerationDegeneration/Inflammation, Vascular 0 (0.0) 0 (0.0) LE 4 cm distalHemorrhage 0 (0.0) 0 (0.0) Mixed inflammation 0 (0.0) 1 (1.5)Edema/Collagen Degeneration 0 (0.0) 1 (2.0) Epidermis, Crust/Pustule,Erosion/ 0 (0.0) 0 (0.0) Ulceration Degeneration/Inflammation, Vascular0 (0.0) 0 (0.0) *The number in parentheses represents the averageseverity score; the total of severity scores of the findings divided bythe number of animals in the group.

Conclusion:

No test-article related irritation effects were observed for thenon-aqueous formulation of bendamustine via the IV route, indicatingthat proper administration of this formulation did not result in anyadverse local reaction. Perivascular administration of the non-aqueousbendamustine formulation, which is primarily related to effects that mayoccur if extravasation should occur, resulted in irritation that wasgenerally comparable to Treanda. Therefore, the non-aqueous formulationof bendamustine described herein is well tolerated, despite the higherconcentration of the smaller infusion volume preparation.

Example 8

The chemical stability of the non-aqueous bendamustine formulation (25mg/ml) indicated in Table 4 (Example 5), when admixed with 50 ml and 100ml of normal saline, was assessed. For each admixture volume, theadmixture solutions were prepared at the expected lowest concentration(corresponding to a 1.0 m² patient dosed at 25 mg/m²) and the highestconcentration (corresponding to a 3.0 m² patient dosed at 120 mg/m²) ofbendamustine HCl in the final admixture. For the 50 ml admixture volume,the tested minimum and maximum concentrations are about 0.5 mg/ml and6.0 mg/ml, respectively. For the 100 ml admixture volume, the testedminimum and maximum concentrations are about 0.25 mg/ml and 3.2 mg/ml,respectively. The chemical stability of Treanda™ was also determined atthe lowest (0.2 mg/ml) and the highest (0.6 mg/ml) admixedconcentrations stated in the label. The chemical stability was monitoredat room temperature at periodic intervals up to 24 hours using avalidated HPLC assay. The results are summarized in Table 9.

TABLE 9 Dilution (Admixed) Stability of Bendamustine Formulations inNormal Saline at Room Temperature Attribute Highest Concentration (3.2mg/ml) Lowest Concentration (0.25 mg/ml) Time Initial 1 hr 3 hrs 6 hrs24 hrs Initial 1 hr 3 hrs 6 hrs 24 hrs Formulation Non AqueousBendamustine Formulation 25 mg/ml admixed with 100 ml normal salineAssay (mg/ml) 3.155 3.090 3.060 3.085 2.895 0.240 0.234 0.229 0.2240.196 Assay (% Initial) 100.0 97.9 97.0 97.8 91.8 100.0 97.5 95.4 93.381.7 Impurity-MCE (%) BLQ BLQ BLQ BLQ BLQ ND ND ND ND ND Impurity-HP1(%) 0.244 0.606 1.237 2.236 6.707 0.525 1.449 3.495 5.529 13.424Impurity-Dimer (%) BLQ BLQ BLQ 0.068 0.158 ND ND ND BLQ 0.063 Singleunknown (%) 0.086 0.061 BLQ BLQ 0.098 ND ND ND ND BLQ Total (%) 0.330.67 1.24 2.30 6.96 0.58 1.45 3.50 5.53 13.49 Attribute HighestConcentration (6.4 mg/ml) Lowest Concentration (0.5 mg/ml) Time Initial1 hr hrs 6 hrs 24 hrs Initial 1 hr 3 hrs 6 hrs 24 hrs Formulation NonAqueous Bendamustine Formulation 25 mg/ml admixed with 50 ml normalsaline Assay (mg/ml) 6.62 6.60 6.60 6.54 6.46 0.475 0.470 0.455 0.4450.394 Assay (% Initial) 100.0 99.7 99.7 98.8 97.8 100.0 98.9 95.8 93.782.9 Impurity-MCE (%) BLQ BLQ BLQ BLQ 0.074 BLQ BLQ BLQ BLQ BLQImpurity-HP1 (%) 0.137 0.265 0.528 0.945 2.967 0.567 1.618 3.719 5.89214.427 Impurity-Dimer (%) BLQ BLQ BLQ 0.050 0.110 BLQ BLQ BLQ 0.0650.115 Single unknown (%) 0.112 0.105 0.086 0.054 0.112 0.057 BLQ ND NDND Total (%) 0.25 0.37 0.61 1.05 3.36 0.67 1.62 3.72 5.96 14.54Attribute Highest Concentration (0.6 mg/ml) Lowest Concentration (0.2mg/ml) Time Initial 1 hr 3 hrs 6 hrs 24 hrs Initial 1 hr 3 hrs 6 hrs 24hrs Formulation Treanda ™ 5 mg/ml admixed with 500 ml normal salineAssay (mg/ml) 0.566 0.558 0.544 0.527 0.454 0.193 0.191 0.185 0.1780.154 Assay (% Initial) 100.0 98.6 96.1 93.1 80.2 100.0 99.0 95.9 92.279.8 Impurity-MCE (%) 0.263 0.261 0.268 0.262 0.263 0.261 0.288 0.2770.250 0.276 Impurity-HP1 (%) 1.250 2.248 4.730 7.287 16.887 1.231 2.2414.770 7.462 17.504 Impurity-Dimer (%) 0.223 0.229 0.269 0.279 0.3260.188 0.185 0.178 0.176 0.252 Single unknown (%) 0.103 0.103 0.101 0.0970.081 0.077 0.079 0.103 0.083 0.066 Total (%) 1.97 2.97 5.50 8.04 17.661.85 3.01 5.41 8.07 18.27 MCE—monochloroethyl derivative; HP1—monohydroxyl bendamustine

As shown in Table 9, Treanda™ when prepared as directed in the label(final concentration between 0.2-0.6 mg/ml) shows total degradation ofabout 5-6% in 3 hours at room temperature (corresponding to the roomtemperature stability claim in the label); monohydroxy bendamustine isthe main degradant. In contrast, the non-aqueous bendamustineformulations admixed in either 50 ml or 100 ml saline show totaldegradation of less than 5-6% over 6 hours at the lowest concentrationstested, indicating that these admixtures are significantly less prone todegradation. This stabilizing effect is particularly pronounced at thehigher concentrations (which are more typical), with chemical stabilityevident for 24 hours at these concentrations. The non-aqueousformulations of bendamustine thus offer better chemical stability thanTreanda™ when admixed into smaller volumes.

Example 9 Solubility and Stability of Bendamustine Hydrochloride inPolar Aprotic Solvents

Equilibrium solubility was determined for solvents including1-methyl-2-pyrrolidone (NMP), 1,3-dimethyl-2-imidazolidinone (DMI),dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), acetone,tetrahydrofuran (THF), dimethylformamide (DMF), and propylene carbonate(PC). The solubility of bendamustine hydrochloride was also determinedfor two solutions, 25 mg/mL niacinamide in DMA and 66% DMA/34% propyleneglycol (PG). A saturated solution of bendamustine hydrochloride was madein triplicate for each solvent or solution and mixed on a Lab-Quake withgentle mixing and low shear for 3 days at room temperature. A sample ofeach suspension was put into a microcentrifuge tube and spun at 10,000rpm for 5 min on an Eppendorf microcentrifuge. The supernatant wasremoved and put into a clean vial. Each solution was diluted with samplesolvent: 50% NMP/50% 0.1% trifluoroacetic acid in water. A reverse phasemethod for bendamustine hydrochloride was used to determine theconcentration of each sample calculated from a standard. Analysis wasperformed within 18 hours of preparation of the diluted sample. Thesolubilities are listed in Table 10 below. Each value is an average ofthree samples.

TABLE 10 Sample* % Purity Assay (mg/mL) NMP 99.1 104.0 DMI 98.5 75.8DMSO 99.5 311.7 DMF 99.6 71.8 66% DMA/34% PG 99.5 110.1 DMA 99.4 56.2 PC98.7 7.7 Niacinamide/DMA 99.2 61.3 *acetone and THF have no measurablesolubility of bendamustine.

The three replicates were combined and mixed well and then pipetted intoamber HPLC vials and placed in stability chambers at 25° C. and 5° C.All the samples were clear and colorless except for the DMI sample whichwas clear and yellow. The 25° C. stability leveled out from about 180days (about 6 months) to about 365 days (about 12 months, about 1 year).At 5° C., all solutions had a purity greater than 90%. The analysis ofstability samples can be seen in the graphs of FIGS. 1 and 2.

TABLE 11 Impurity profile of certain liquid formulations of BendamustineHCl after storage at 5° C. for about 12 months BM1 DCE HP1 dimer PG-1PG-2 Formulation (Area %) (Area %) (Area %) (Area %) (Area %)Niacinamide/ 1.40 0.08 0.06 ND ND DMA DMA 1.10 0.08 0.05 ND ND 66% DMA/0.12 0.08 0.06 1.09 0.27 34% PG DMF 0.07 0.11 0.07 ND ND NMP 0.90 0.10ND ND ND DMSO 0.04 0.38 0.70 ND ND ND = not detectedAnalysis conducted using reverse phase HPLC with 50% NMP/50% 0.1%trifluoroacetic acid in water as the running solvent.

As can be seen in FIG. 3, bendamustine (BM1) in 99% propylene glycoldegrades significantly when stored at 25° C. for less than 100 days.After storage at 5° C. for about 365 days, the purity of thebendamustine is about 80% or less.

Pharmacokinetic Study of Formulations in Monkey

4 fasted (18 to 23 hr), drug-naive male cynomolgus monkeys consecutivelyreceived single 3-mg/kg bolus intravenous doses of bendamustinehydrochloride prepared from 3 different formulations. The formulationsevaluated in the study included: 1) TREANDA (lyophilized mixture ofbendamustine hydrochloride and mannitol; 25 mg (bendamustinehydrochloride) vials; 2) a 66% dimethylacetamide (DMA)/34% propyleneglycol (PG) (w/w) solution (90 mg (bendamustine hydrochloride)/mLstock); and 3) a 100% DMA solution (45 mg (bendamustinehydrochloride)/mL stock). The lyophilized powder and stock solutions ofbendamustine hydrochloride were constituted or diluted with 0.9% saline,as appropriate, to give solutions of 3 mg bendamustine hydrochloride/ml,just prior to dose administration. The resulting solutions wereadministered as a bolus via a saphenous vein at a fixed volume of 1.0mL/kg. There was at least a 7-day washout period separating successivedoses. During all 3 phases of dosing, blood samples for pharmacokineticprofiling of bendamustine and its 2 active circulating metabolites,γ-hydroxybendamustine (M3) and N-des-methylbendamustine (M4), werecollected via a femoral vein immediately prior to dosing and atpreselected timepoints through 12 hr postdose. Concentrations ofbendamustine, M3 and M4 in plasma samples were determined using avalidated high-performance liquid chromatography method with tandem massspectrometric detection (LC-MS/MS) as follows. Bendamustine and the M3and M4 metabolites are extracted from plasma by protein precipitationusing acetonitrile. After the extraction, the aliquoted sample isacidified with 1% formic acid and bendamustine with an added carbon inthe carboxylic acid chain is added as an internal standard. The samplesare evaporated to dryness and the residue is reconstituted with anacetonitrile/water/formic acid/ammonium formate mixture. The sample isinjected into an HPLC system with LC/MS/MS detection using a PhenomenexSynergi Max-RP column with an acetonitrile/water/formic acid/ammoniumformate mobile phase. Pharmacokinetic analyses were performed usingnoncompartmental methods.

After single bolus intravenous doses of bendamustine hydrochloride tomale cynomolgus monkeys, the shapes of the mean plasmaconcentration-versus-time profiles of bendamustine were similar in eachof the 3 formulations (See FIG. 4). In all cases, the highest observedplasma levels of bendamustine were achieved at 0.083 hr postdose (ie,the first sampling time after dose administration) and subsequentremoval of the compound from plasma occurred in a bi-phasic manner thatwas characterized by an initial rapid distribution phase and a somewhatslower terminal phase of drug elimination. The harmonic mean t_(1/2) ofthe terminal phase was approximately 0.6 hr for each formulation (SeeTable 12).

In addition to the similarities in the shapes of the mean plasmaconcentration-versus-time profiles, the 3 formulations were also similarwith respect to bendamustine systemic exposure (i.e., C_(max) and AUC).Specifically, the respective mean values of C_(max) and AUC_(0-∞) forbendamustine were 6037 ng/mL and 2314 ng·hr/mL for the TREANDAformulation, 7380 ng/mL and 2854 ng·hr/mL for the 66% DMA/34% PGformulation and 6209 ng/mL and 2372 ng·hr/mL for the 100% DMAformulation. Plasma clearance (CL) and volume of distribution (V_(z) andV_(ss)) for bendamustine were also comparable between each of the 3formulations (See Table 12). In Table 12, t_(max), hr is given as Median[range], t_(1/2), hr is given as the Harmonic Mean, λ_(z), hr⁻¹ is theslope of line in elimination phase used to calculate half-life, andMRT_(0-∞) is the mean residence time.

In summary, the pharmacokinetic profiles of bendamustine, M3 and M4 forthe 2 liquid formulations of bendamustine hydrochloride werequalitatively and quantitatively similar to those obtained for theTREANDA formulation after single bolus intravenous doses to monkeys.

Table 12 shows the mean+/−Standard Deviation pharmacokinetic parametersof bendamustine in male Cynomolgus monkeys (N=4) administered single 3mg/kg bolus intravenous doses of bendamustine hydrochloride in the threedifferent formulations.

TABLE 12 Formulation Parameter TREANDA 66% DMA/34% PG 100% DMA C₀, ng/mL8664 ± 3841 10716 ± 2033  8956 ± 1965 C_(max), ng/mL 6037 ± 2456 7380 ±1170 6209 ± 1300 t_(max), hr 0.083 [0.083 0.083 [0.083 0.083 [0.083 forall] for all] for all] AUC_(0-t), ng · hr/mL 2313 ± 800  2853 ± 398 2371 ± 535  AUC_(0-∞), ng · hr/mL 2314 ± 800  2854 ± 398  2372 ± 535 λ_(z), hr⁻¹ 1.220 ± 0.111 1.295 ± 0.108 1.092 ± 0.219 t_(1/2), hr 0.570.54 0.63 CL, L/hr/kg 1.27 ± 0.40 0.96 ± 0.14 1.18 ± 0.27 V_(z), L/kg1.04 ± 0.36 0.74 ± 0.05 1.17 ± 0.44 V_(ss), L/kg 0.34 ± 0.11 0.26 ± 0.050.30 ± 0.04 MRT_(0-∞), hr 0.26 ± 0.02 0.27 ± 0.02 0.26 ± 0.03

In-Use Studies of Formulations

Admixtures in 0.9% sodium chloride (500 mL bag) were prepared at a highdose (360 mg bendamustine hydrochloride) and purity was determined overtime at room temperature for up to 8 hours using HPLC, using a ZorbaxBonus-RP column with a gradient from 93% 0.1% trifluoroacetic acid inwater (Mobile Phase A)/7% 0.1% trifluoroacetic acid in acetonitrile(Mobile Phase B) to 10% Mobile Phase A/90% Mobile Phase B.

The 66% DMA/34% PG formulation had a concentration of bendamustinehydrochloride of 90 mg/g, so 4 mL was injected into a 500 mL bag ofsaline, inverted 10 times and sampled at room temperature for 8 hours.After 8 hours the purity was 95.4%. This is within the labelrequirements for dosing Treanda. This formulation of the presentinvention could be used for up to 8 hours at room temperature. By way ofcontrast, reconstituted Treanda can only be stored at room temperaturefor up to 3 hours.

The 100% DMA formulation had a concentration of 45 mg/g, so 8 mL wasinjected into a 500 mL bag of saline, inverted 10 times, and sampled atroom temperature for 4 hours. After 4 hours the purity was 97.9%. Thisformulation of the present invention could be used for more than 4 hoursat room temperature.

The comparative Treanda admixture purity was 95.0% after 4 hours at 25°C.

Example 10

Preparation of a Bendamustine Composition with Sodium Sulfobutyl Etherβ-Cyclodextrin and Chondroitin

6 mg of bendamustine hydrochloride were dissolved in 1 mL of 20% w/wsolution of sodium sulfobutyl ether β-cyclodextrin. The solution wasincubated at 20° C. for 15 minutes on ultrasonic bath and mixed with 1mL of 25% solution of chondroitin sulfate.

Example 11

Preparation of a Bendamustine Composition with Sodium Sulfobutyl Etherβ-Cyclodextrin and Poly(Sulfonylbutylo)Cellulose

6 mg of bendamustine hydrochloride are dissolved in 1 ml of 30% w/wsolution of sodium sulfobutyl ether β-cyclodextrin. The solution ispreincubated at 10° C. for 15 minutes on ultrasonic bath. After that thesolution is mixed with 1 ml of 2% solution ofpoly(sulfonylbutylo)cellulose sodium salt. The sample is incubated onultrasonic bath for 30 minutes at 10° C.

Example 12

Preparation of a Bendamustine Composition with Sodium Sulfobutyl Etherβ-Cyclodextrin and Hyaluronic Acid

6 mg of bendamustine hydrochloride were dissolved in 1 mL of 30% w/wsolution of sodium sulfobutyl ether β-cyclodextrin. The solution waspreincubated at 10° C. for 15 minutes on ultrasonic bath. After that thesolution was mixed with 1 mL of 0.1% solution of hyaluronic acid sodiumsalt. The sample was incubated on ultrasonic bath for 30 minutes at 10°C.

Example 13

Preparation of a Bendamustine Composition with Sodium Sulfobutyl Etherβ-Cyclodextrin and Dextran

6 mg of bendamustine hydrochloride were dissolved in 1 mL of 20% w/wsolution of sodium sulfobutyl ether β-cyclodextrin. The solution wasincubated at 20° C. for 15 minutes on ultrasonic bath and then mixedwith 1 mL of 50% solution of dextran 40 (MW 40000) and sonicated foranother 15 minutes.

Example 14

Preparation of a Bendamustine Composition with2-Hydroxypropyl-β-Cyclodextrin and Dextran

6 mg of bendamustine hydrochloride were dissolved in 1 mL of 20% w/wsolution of 2-hydroxypropyl-β-cyclodextrin. The solution was incubatedat 20° C. for 15 minutes on ultrasonic bath and then mixed with 1 mL of50% solution of dextran 40 (MW 40000) and was sonicated for another 15minutes.

Example 15

Preparation of a Bendamustine Composition with Sodium Sulfobutyl Etherβ-Cyclodextrin and Quaternized Cellulose

2 mg of quaternized cellulose (hydrochloride salt) were dissolved in 1mL of water. After 4 hours of preincubation on an ultrasonic bath (atroom temperature), the solution was mixed with a solution of 6 mg ofbendamustine hydrochloride in 1 mL of 20% w/w solution of sodiumsulfobutyl ether β-cyclodextrin. The solution was well mixed andincubated for 15 minutes on ultrasonic bath.

Example 16

Preparation of a Bendamustine Composition with Sodium Sulfobutyl Etherβ-Cyclodextrin and Polyvinylpyrrolidone

6 mg of bendamustine hydrochloride were dissolved in 1 mL of 30% w/wsolution of sodium sulfobutyl ether β-cyclodextrin. The solution wasincubated at 20° C. for 15 minutes on an ultrasonic bath and then mixedwith 1 mL of 20% solution of PVP (MW=10000) and sonicated for 20minutes.

Example 17

Preparation of a Bendamustine Composition with Sodium Sulfobutyl Etherβ-Cyclodextrin and Cetylpyridinium Chloride

6 mg of bendamustine hydrochloride and 8.5 mg of mannitol were dissolvedin 0.8 g of 50% w/w solution of sodium sulfobutyl ether β-cyclodextrin.The solution was incubated at 20° C. for 15 minutes on ultrasonic bathand then mixed with 0.2 mL of 2.5% solution of cetylpyridinium chloride.

Example 18

Preparation of Bendamustine Composition with Sodium Sulfobutyl Etherβ-Cyclodextrin and PI2080 Preparation of PI2080

Polyethyleneimine (PEI, MW 2000) was purchased from Aldrich.Poly(ethylene glycol) monomethyl ether (PEG, MW 8500) was purchased fromPolymer Sources Inc. PI2080, a conjugate of PEG and PEI, was preparedfollowing the procedure described by Vinogradov S. V. et al. inBioconjugate Chem. 1998, 9, 805-812.4 g of PEG was reacted with1,1′-carbonyldiimidazole in 20 mL anhydrous acetonitrile. The product ofthe reaction was dialysed twice against water using SpectraPor 3membrane, MWCO 3500, and freeze-dried. The freeze-dried material wasdissolved in 32 mL of methanol, mixed with 2.9 g of PEI and incubatedfor 24 hours at 25° C. The product was dialysed twice against waterusing SpectraPor 3 membrane, MWCO 3500, and the product PI2080 wasfreeze-dried.

Example 19

Preparation of Bendamustine Composition with Sodium Sulfobutyl Etherβ-Cyclodextrin and PI2080

2.5 mg of bendamustine hydrochloride and 4.3 mg of mannitol weredissolved in 0.8 g of 50% w/w solution of sodium sulfobutyl etherβ-cyclodextrin. The solution was incubated at 20° C. for 15 minutes onultrasonic bath and then mixed with 0.2 mL of 5% solution of PI2080.

Example 20

Preparation of Bendamustine Composition with Sodium Sulfobutyl Etherβ-Cyclodextrin and with Protamine Sulphate

2.5 mg of bendamustine hydrochloride and 4.3 mg of mannitol weredissolved in 0.800 g of 50% w/w solution of Sodium sulfobutyl etherβ-cyclodextrin. Solution was shaken at 20° C. for 90 minutes and thenincubated for 30 minutes in an ultrasonic bath. Then the mixture wastransferred into a suspension of 30 mg of protamine sulfate in 0.163 gof water and vigorously mixed for 15 minutes.

Example 21

Pharmacokinetics of Bendamustine Dosed to Rats in Composition withSodium Sulfobutyl Ether β-Cyclodextrin and Cetylpyridinium Chloride

The Tested Compositions:

Control: 6 mg/mL bendamustine hydrochloride, 10.2 mg/mL of mannitol in0.9% NaCl; dose of 20 mg/kg

Inventive Composition: 5 mg/g bendamustine hydrochloride, 40% w/w sodiumsulfobutyl ether β-cyclodextrin, 0.5% cetylpyridinium chloride, 8.5 mg/gmannitol in water (produced following the procedure of Example 17); doseof 20 mg/kg.

We claim:
 1. A method of treating chronic lymphocytic leukemia orindolent B cell non-Hodgkin's lymphoma in a subject comprisingparenterally administering to the subject a volume of about 100 mL orless of a liquid composition comprising: bendamustine; a chargedcyclopolysaccharide; and a parenterally acceptable diluent; over aperiod of less than or equal to about 15 minutes.
 2. The method of claim1, wherein the subject is human.
 3. The method of claim 1, wherein theconcentration of the bendamustine or pharmaceutically acceptable saltthereof is from about 0.05 to about 12.5 mg/mL.
 4. The method of claim1, wherein the concentration of the bendamustine or pharmaceuticallyacceptable salt thereof is from about 0.1 to about 6.0 mg/mL.
 5. Themethod of claim 1, wherein the concentration of the bendamustine orpharmaceutically acceptable salt thereof is from about 0.05 to about 3.2mg/mL.
 6. The method of claim 1, wherein the concentration of thebendamustine or pharmaceutically acceptable salt thereof is from about0.5 to about 5.6 mg/mL.
 7. The method of claim 1, wherein the volumeadministered is about 100 ml, about 50 mL, about 30 mL, or about 5 mL.8. The method of claim 1, wherein the volume administered is about 50mL.
 9. The method of claim 1, wherein the bendamustine is administeredto treat chronic lymphocytic leukemia.
 10. The method of claim 9,wherein the composition is administered intravenously in a volume ofabout 50 mL in about 10 minutes or less on days 1 and 2 of a 28 daycycle.
 11. The method of claim 10, wherein the composition isadministered in about 10 minutes.
 12. The method of claim 10, whereinthe composition is administered for up to 6 cycles.
 13. The method ofclaim 9, wherein the volume of the composition administered to thesubject provides a bendamustine dosage amount ranging from about 25mg/m² to about 100 mg/m² to the subject.
 14. The method of claim 13,wherein the composition provides a bendamustine dosage of about 100mg/m².
 15. The method of claim 9, wherein the liquid compositioncomprises from about 1.85 mg/mL to about 4.84 mg/mL of bendamustine or apharmaceutically acceptable salt thereof.
 16. The method of claim 1,wherein the bendamustine is administered to treat indolent B cellnon-Hodgkin's lymphoma.
 17. The method of claim 16, wherein thecomposition is administered intravenously in a volume of about 50 mL inabout 10 minutes or less on days 1 and 2 of a 21 day cycle.
 18. Themethod of claim 17, wherein the composition is administered in about 10minutes.
 19. The method of claim 17, wherein the composition isadministered for up to 8 cycles.
 20. The method of claim 16, wherein thevolume of the composition administered to the subject provides abendamustine dosage amount ranging from about 60 mg/m² to about 120mg/m² to the subject.
 21. The method of claim 20, wherein thecomposition provides a bendamustine dosage of about 120 mg/m².
 22. Themethod of claim 16, wherein the liquid composition comprises from about2.19 mg/mL to about 5.59 mg/mL of bendamustine or a pharmaceuticallyacceptable salt thereof.
 23. The method of claim 1, wherein thecyclopolysaccharide is a beta-cyclodextrin.
 24. The method of claim 1,wherein the charged cyclopolysaccaride has one or more anionic groups.25. The method of claim 24, wherein the anionic group is a sulfate,sulfonyl, or carbonyl group.
 26. The method of claim 24, wherein theanionic group is a sulfate or sulfonyl group.
 27. The method of claim24, wherein the cyclopolysaccharide is sulfobutyl etherbeta-cyclodextrin.
 28. The method of claim 1, wherein the proportion ofbendamustine to cyclopolysaccaride is between about 1:12,500 and about1:25, by weight.
 29. The method of claim 1, wherein the compositionfurther comprises a stabilizing agent.
 30. The method of claim 29,wherein the stabilizing agent is a polypeptide comprising from about 5to about 50 amino acids, wherein at least about 50% of the amino acidshave a positive charge.
 31. The method of claim 29, wherein thestabilizing agent is a polypeptide comprising between about 6 and about20 amino acids, wherein at least about 50% of the amino acids have apositive charge.
 32. The method of claim 30, wherein the polypeptidecomprises at least one block sequence of 4 arginines.
 33. The method ofclaim 32, wherein the stabilizing agent is polyarginine.
 34. The methodof claim 30, wherein the stabilizing agent is low molecular weightprotamine.
 35. The method of claim 1, wherein the charged group on thecyclopolysaccharide is a cationic group.
 36. The composition of claim35, wherein the cationic group is a quaternary ammonium group.
 37. Themethod of claim 1, wherein the bendamustine is present as thehydrochloride salt.